Cellular signaling has become a major research theme in biology and medicine over the past twenty years. The complex pathways and protein components in signal transduction are emerging with increasing clarity. Over the last 15 years, the protein kinases, such as the protein tyrosine kinases, have been identified as key players in cellular regulation. They are involved in immune, endocrine, and nervous system physiology and pathology and thought to be important in the development of many cancers. As such they serve as drug targets for many different diseases. Members of protein kinase family include for example c-Jun N-terminal kinase or Glycogen Synthase Kinase 3 (GSK3).
C-Jun N-Terminal kinase (JNK) is a member of the MAP Kinase family that includes the extracellular regulated kinases (ERKs) and p38 kinases. It is a serine/threonine kinase that phosphorylates c-Jun, a transcription factor activator protein-1 (AP-1) component. AP-1 regulates the transcription of several genes including inflammatory enzymes (COX-2), matrix metalloproteinases (MMP-13), cytokines (TNF), growth factors (VEGF) and immunoglobulins. Three JNK isoforms, JNK-1, -2 and -3, have been identified in humans and they appear to mediate critical phosphorylation events involved in the regulation of apoptosis and the immune response.
In a publication of Xie X et al, (Structure 6 (8) p. 983-991 (1998)) it has been suggested that activation of stress-activated signal transduction pathways are required for neuronal apoptosis induced by NGF withdrawal in rat PC-12 and superior cervical ganglia (SCG) sympathetic neuronal cells. Inhibition of specific kinases, namely MAP kinase kinase 3 (MKK3) and MAP kinase kinase 4 (MKK4), or c-Jun (part of the MKK-4 cascade) may be sufficient to block apoptosis (see also Kumagae Y et al, in Brain Res, 67(1), 10-17 (1999) and Yang DD et al in Nature, 389 p. 865-870 (1997)).
It has been reported that the JNK signalling pathway is implicated in cell proliferation and could play an important role in autoimmune diseases (Yang et al, Immunity 9, 575-585 (1998); Sabapathy et al. Current Biology 3, 116-125 (1999)) which are mediated by T-cell activation and proliferation.
One of the first compounds that inhibits the JNK pathway is Cephalon's CEP-1347 which was found to be neuroprotective in a number of in vivo models of neurodegenerative disease. Several compounds are reported in the patent literature to inhibit JNKs. Hoffmann-La Roche claimed 4-heteroaryl, 4-arylindolinones and annulated indolinones (WO 0035921, WO 0035909 and WO 0035906). Vertex Pharmaceuticals disclosed oxime derivatives as a JNK3 inhibitor (WO 0064872). Applied Research Systems has disclosed benzazole derivatives (EP 1110957) as JNK inhibitors.
Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase for which two isoforms, α and β, have been identified (Woodgett et al. Trends Biochem. Sci., 16 p. 177-81 (1991)). Both GSK3 isoforms are constitutively active in resting cells. GSK3 was originally identified as a kinase that inhibits glycogen synthase by direct phosphorylation. Upon insulin activation, GSK3 is inactivated, thereby allowing the activation of glycogen synthase and possibly other insulin-dependent events, such glucose transport. Subsequently, it has been shown that GSK3 activity is also inactivated by other growth factors that, like insulin, signal through receptor tyrosine kinases (RTKs). Examples of such signalling molecules include IGF-1 and EGF (Saito et al. Biochem. J., 303 p. 27-31 (1994), Welsh et al., Biochem. J., 294 p. 625-29 (1993) and Cross et al., Biochem. J., 303 p. 21-26 (1994)). GSK3 beta activity is regulated by serine (inhibitory) and tyrosine (stimulatory) phosphorylation, by protein complex formation, and by its intracellular localization. GSK3 beta phosphorylates and thereby regulates the functions of many metabolic, signalling and structural proteins (Carol Grimes, Richard Jope, Prog. Neurobiol. 65(4) p. 391-426 (2001)). Notable among the signalling proteins regulated by GSK3 beta are the many transcription factors, including activator protein-1 cells, Myc, beta-catenin, CCAAT/enhancer binding protein, and NFkappaB.
Agents that inhibit GSK3 activity are useful in the treatment of disorders that are mediated by GSK3 activity. In addition, inhibition of GSK3 mimics the activation of growth factor signalling pathways and consequently GSK3 inhibitors are useful in the treatment of diseases in which such pathways are insufficiently active. Examples of diseases that can be treated with GSK3 inhibitors, such as diabetes, neurodegenerative diseases (e.g. Alzheimer's disease), inflammatory diseases, ischemia and cancer are described below.
In the patent literature, several GSK3 inhibitors have already been disclosed (WO 02/20495, Chiron Corporation; WO 02/10141, Pfizer Products Inc.; WO 02/22608, Vertex Pharmaceuticals Inc.).
Diabetes mellitus is a serious metabolic disease that is defined by the presence of chemically elevated levels of blood glucose (hyperglycemia). The term diabetes mellitus encompasses several different hyperglycemic states. These states include Type 1 (insulin-dependent diabetes mellitus or IDDM) and Type 2 (non-insulin dependent diabetes mellitus or NIDDM) diabetes. The hyperglycemia present in individuals with Type 1 diabetes is associated with deficient, reduced, or nonexistent levels of insulin that are insufficient to maintain blood glucose levels within the physiological range. Conventionally, Type 1 diabetes is treated by administration of replacement doses of insulin, generally by a parenteral route.
Type 2 diabetes is an increasingly prevalent disease of aging. It is initially characterized by decreased sensitivity to insulin and a compensatory elevation in circulating insulin concentrations, the latter of which is required to maintain normal blood glucose levels. As described above, GSK3 inhibition stimulates insulin-dependent processes and is consequently useful in the treatment of type 2 diabetes. Recent data obtained using lithium salts provides evidence for this notion. The lithium ion has recently been reported to inhibit GSK3 activity (Peter Klein, Douglas Melton PNAS 93 p. 8455-9 (1996)). However, lithium has not been widely accepted for use in the inhibition of GSK3 activity, possibly because of its documented effects on molecular targets other than GSK3. The purine analog 5-iodotubercidin, also a GSK3 inhibitor, likewise stimulates glycogen synthesis and antagonizes inactivation of glycogen synthase by glucagon and vasopressin in rat liver cells (Fluckiger-Isler et al., Biochem. J. 292 p. 85-91 (1993) and Massillon et al., Biochem. J. 299 p. 123-8 (1994)). However, this compound has also been shown to inhibit other serine/threonine and tyrosine kinases (Biochem. J. 299 p. 123-8 (1994)).
GSK(3 is also involved in biological pathways relating to Alzheimer's disease (AD). The characteristic pathological features of AD are extracellular plaques of an abnormally processed form of the amyloid precursor protein (APP), so-called β-amyloid peptide (β-AP) and the development of intracellular neurofibrillary tangles containing paired helical filaments (PHF) that consists largely of hyperphosphorylated tau protein. GSK3 is one of a number of a number of kinases that have been found to phosphorylate tau protein in vitro on the abnormal sites characteristic of PHF tau, and is the only kinase also demonstrated to do this in living cells and in animals (Lovestone et al., Current Biology 4 p. 1077-86 (1994) and Brownlees et al., Neuroreport 8 p. 3251-55 (1997)). Furthermore, the GSK3 kinase inhibitor, LiCl, blocks tau hyperphosphorylation in cells (Stambolic et al., Current Biology 6 p. 1664-8 (1996)). Thus GSK3 activity may contribute to the generation of neurofibrillary tangles and consequently to disease progression. Recently it has been shown that GSK3b associates with another key protein in AD pathogenesis, presenillin 1 (PS1) (Takashima et al., PNAS 95 p. 9637-41 (1998)). Mutations in the PS1 gene lead to increased production of β-AP, but the authors also demonstrate that the mutant PS1 proteins bind more tightly to GSK3β and potentiate the phosphorylation of tau, which is bound to the same region of PS1. Interestingly it has also been shown that another GSK3 substrate, β-catenin, binds to PS1 (Zhang et al., Nature 395 p. 698-702 (1998)). Cytosolic β-catenin is targeted for degradation upon phosphorylation by GSK3 and reduced β-catenin activity is associated with increased sensitivity of neuronal cells to β-AP induced neuronal apoptosis. Consequently, increased association of GSKβ with mutant PS1 may account for the reduced levels of β-catenin that have been observed in the brains of PS1-mutant AD patients and to the disease related increase in neuronal cell-death. Consistent with these observations, it has been shown that injection of GSK3 antisense but not sense, blocks the pathological effects of β-AP on neurons in vitro, resulting in a 24 hr delay in the onset of cell death (Takashima et al., PNAS 90 p. 7789-93 (1993)). In these latter studies, the effects on cell-death are preceded (within 3-6 hours of β-AP administration) by a doubling of intracellular GSK3 activity, suggesting that genetic mechanisms may increase GSK3 activity. Further evidence for a role for GSK3 in AD is provided by the observation that the protein expression level (but, in this case, not specific activity) of GSK3 is increased by 50% in postsynaptosomal supernatants of AD vs. normal brain tissue (Pei et al., J Neuropathol. Exp. 56 p. 70-78 (1997)). Thus, it is believed that specific inhibitors of GSK3 will act to slow the progression of Alzheimer's Disease.
It has also been described an involvement of GSK3 activity in the etiology of bipolar disorder. In support of this notion it was recently shown that valproate, another drug commonly used in the treatment of said disease, is also a GSK3 inhibitor (Chen et al. J Neurochemistry 72 p. 1327-30 (1999)). One mechanism by which lithium and other GSK3 inhibitors may act to treat bipolar disorder is to increase the survival of neurons subjected to aberrantly high levels of excitation induced by the neurotransmitter, glutamate (Nonaka et al, PNAS 95 p. 2642-47 (1998).
Glutamate-induced neuronal excitotoxicity is also believed to be a major cause of neurodegeneration associated with acute damage such as in cerebral ischemia, traumatic brain injury and bacterial infection. Furthermore, it is believed that excessive glutamate signalling is a factor in the chronic neuronal damage seen in diseases such as Alzheimer's, Huntingdon's, Parkinson's, AIDS associated dementia, amyotrophic lateral sclerosis (AML) and multiple sclerosis (MS) (Thomas et al., J. Am. Geriatr. Soc. 43 p. 1279-89 (1995)). Consequently, GSK3 inhibitors are believed to be a useful treatment in these and other neurodegenerative disorders.
Sasaki et al. disclosed that GSK3 beta may have a role in ischemic neuronal cell death (Sasaki C. et al., Neurol. Res. 23(6) p. 588-92 (2001). Cross et al. described selective small-molecule inhibitors of glycogen synthase kinase-3 activity protecting primary neurones from death (Cross et al., Journal of Neurochemistry 77 p. 94-102 (2001)).
It has also been reported that debromohymenialdisine (DBH), considered as inhibitors of GSK3, exhibit anti-inflammatory activity in a model of adjuvant-induced arthritis in the rat. (A. Ali et al., American Chemical Society p. A-N (December 2000)).